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ChIP assays were performed using human HeLa cells. A titration of the antibody consisting of 1, 2, 5 and 10 ug per ChIP experiment was analysed. IgG (1 ug/IP) was used as negative IP control. QPCR was performed with primers for the GAPDH and EIF4A2 promoters, used as positive controls, and for the HBB promoter and the Sat2 satellite repeat, used as negative controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

ChIP was performed on sheared chromatin from 4 million HeLa cells. The figure shows the peak distribution along the complete sequence and a 1.5 mb region of the X chromosome and in two regions surrounding the GAPDH and EIF4A2 positive control genes.

Western Blot (Cell lysate) analysis of (1) 25 ug whole cell extracts of Hela cells, (2) 15 ug histone extracts of Hela cells.

Immunofluorescent staining of Hela cell line with antibody followed by an anti-rabbit antibody conjugated to Alexa488 (left). The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings (right).

ELISA is a quantitative method used to determine the titer of the antibody using a serial dilution of antibody against Histone H3 (K4ac) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:197500.

Cross reactivity test using the Histone H3 (K4ac) antibody.
Dot Blot analysis was performed with peptides containing other histone H3 and H4 modifications and the unmodified H3K4 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:5000. The figure shows a high specificity of the antibody for the modification of interest.